Naturally occurring interferons (IFNs) are species-specific proteins, often glycoproteins, produced by various cells upon induction with viruses, double stranded RNAs, other polynucleotides, antigens and mitogens. Interferons exhibit multiple biological activities such as antiviral, antiproliferative, immunomodulatory and anticellular functions. At least three distinct types of human interferons have been identified and characterized in terms of their anti-viral, anti-growth and activation of natural killer cell (NK) activities. They are produced by leukocytes, lymphocytes, fibroblasts and the immune system and are classified as .alpha., .beta. and .gamma. interferons. These are reported to be different proteins coded for by distinct structural genes.
Native human .beta.-interferon (HuIFN-.beta.) is generally produced by superinducing human fibroblast cultures with poly-IC (poly-riboinosinic acid and polyribocytidylic acid) and isolating and purifying the HuIFN-.beta. thus produced by chromatographic and electrophoretic techniques. Proteins or polypeptides which exhibit native .beta.-interferon properties may also be produced using recombinant DNA technology by extracting poly-A-rich 12S messenger RNA from virally induced human cells, synthesizing double-stranded c-DNA using the m-DNA as a template, introducing the c-DNA into an appropriate cloning vector, transforming suitable microorganisms with the vector, harvesting the bacteria and extracting the HIFN-.beta. therefrom. Nagola, S. et al., Nature, 284:316 (1980); Goeddel, D. V. et al., Nature, 287:411 (1980); Yelverton, E. et al., Nuc. Acid Res., 9:731 (1981); Streuli, M. et al., Proc. Nat'l Acad. Sci. (U.S.), 78:2848 (1981); European Pat. Application No. 28033, published May 6, 1981; 321134, published July 15, 1981; 34307 published Aug. 26, 1981; and Belgian Patent No. 837379, issued July 1, 1981 described various currently used methods for the production of .beta.-interferon employing recombinant DNA techniques. The expressed proteins or polypeptides have been purified and tested and have been found to exhibit properties similar to those of native IFNs. Bacterially produced IFNs thus appear to have potential therapeutic use as antiviral and anti-tumor agents and the production of IFNs by such bacterial fermentations is expected to yield sufficiently large quantities of IFN at a relatively low cost for clinical testing.
Further, HuIFN-.beta. genes have been altered by, for example, oligonucleotide-directed mutagenesis to produce IFN-.beta. protein analogs thereof, such as the human recombinant cysteine-depleted or cysteine-replaced interferon-.beta. analogs (muteins) disclosed in U.S. Pat. No. 4,588,585 issued May 13, 1986 to Mark et al. Specifically disclosed in that patent is the recombinant IFN-.beta. mutein wherein the cysteine at position 17 is replaced by a neutral amino acid such as serine. The latter IFN-.beta. mutein is IFN-.beta..sub.ser17.
Procedures for recovering and purifying bacterially produced IFNs are described in U.S. Pat. Nos. 4,450,103; 4,315,852; 4,343,735; and 4,343,736; and Derynck et al., Nature (1980) 287:193-197 and Scandella and Kornberg, Biochemistry, 10:4447 (1971).
E. coli expressed recombinant IFN-.beta. and analogs thereof are insoluble in solutions which are at a pH range of 6 to 9. Therefore, various processes and additives have been devised to solubilize these proteins. Several methods currently available for the preparation, recovery and purification of bacterially produced proteins are listed immediately below.
U.S. Pat. No. 4,315,852 to Leibowitz et al., describes a method for the acid extraction of leukocyte interferon from bacterial cells and neutralization of the extractant to obtain the interferon.
U.S. Pat. No. 4,343,735 to Menge et al., teaches a process for the purification of interferon by partitioning it in an aqueous multi-phase system in the presence of ion exchangers which are soluble in the system and are derivatives of polyethers.
U.S. Pat. No. 4,343,736 to Uemura et al. discloses a method for recovering interferon by absorption of water-insolubilized heparin and then eluting the interferon with an aqueous solution of an inorganic salt and chondroitin sulfate.
U.S. Pat. No. 4,289,689 to Friesen et al. discloses how to recover and purify human native .beta.-interferon by use of affinity chromatography and high pressure liquid chromatography.
U.S. Pat. No. 4,364,863 to Leibowitz et al. describes a method of extracting fibroblast interferon from bacteria using a low pH followed by a high pH extraction procedure.
U.S. Pat. No. 4,450,103 to Konrad et al. discloses solubilizing the protein in an aqueous medium with an appropriate solubilizing agent, extracting the protein from the aqueous medium with 2-butanol or 2-methyl-2-butanol, and precipitating the protein from the alcohol phase.
U.S. Pat. No. 4,530,787 to Shaked et al. describes a process for oxidizing recombinant proteins such as IFN-.beta. selectively and stoichiometrically using o-iodosobenzoic acid to ensure that the protein will be functionally equivalent to its native counterpart.
Many heterologous proteins are precipitated intracellularly in the form of refractile or inclusion bodies which appear as bright spots visible within the enclosure of the cell under a phase contrast microscope at magnifications down to 1000 fold. See e.g., Miller et al., Science (1982) 215:687-690; Cheng, Biochem. Biophys. Res. Comm., (1983) 111:104-111; Becker et al., Biotech. Advs. (1983) 1:247-261; Kleid et al., ch. 25 in Developments in Industrial Microbiology, Vol. # 25, pp. 317-325 (Society for Industrial Microbiology, Arlington, Va., 1984); Marston et al., Bio/Technology (Sept., 1984), pp. 800-804.
Purification and activity assurance of precipitated heterologous proteins is also described by U.S. Pat. Nos. 4,511,502; 4,511,503; 4,512,922; 4,599,127 and 4,518,526; and EP No. 114,506.
Wang et al., J. Parenteral. Drug Assoc., 34, 452-462 (1980) provides a review of excipients and pHs for parenteral products used in the U.S. Table I therein under section II entitled "Solubilizers, Wetting Agents or Emulsifiers" lists among other excipients polyethylene glycol 300 and glycerin.
U.S. Pat. No. 4,647,454 to Cymbalista et al. discloses a method of stabilizing human fibroblast interferon with polyvinyl pyrrolidone.
U.S. Pat. No. 4,460,574 to Yabrov discloses a pharmaceutical composition comprising native human .alpha.- and .beta.-interferons used for rectal or urogenital treatment of human interferon-sensitive diseases.
U.S. Pat. No. 4,462,940 to Hanisch et al. ('940 patent) discloses a process for formulating interferon by mixing the interferon and a protein stabilizer, such as normal serum albumin, at a pH of about 10.5 to 12.5 for 5 minutes and then adjusting the pH to 7.5 to obtain a soluble mixture.
Copending, commonly owned, U.S. application Ser. No. 775,751, filed Sept. 13, 1985 entitled "An Improved Formulation for Lipophilic Proteins" outlines a high pH and a low pH process for removing and purifying lipophilic recombinant proteins, such as human IFN-.beta. and interleukin-2, from host strains to yield a protein preparation which may be formulated into a stable pharmaceutical composition. Said composition carrying a therapeutically effective amount of the biologically active lipophilic protein dissolved in a not-toxic, inert, therapeutically compatible aqueous-based carrier medium at a pH of 6.8 to 7.8 also contains a stabilizer for the protein, such as human serum albumin, human serum albumin and dextrose, or human plasma protein fraction.
Copending, commonly owned, U.S. application Ser. No. 780,551, filed Sept. 26, 1985, entitled "Stable Formulation of Biologically Active Proteins for Parenteral Injection," discloses pharmaceutical compositions containing IFN-.beta. or interleukin-2 dissolved in a stable carrier medium at pH 7.0 to 8.0 stabilized with sodium laurate.
Copending, commonly owned, U.S. application Ser. No. 749,955 filed June 26, 1985, and 866,459 filed May 21, 1986 (Katre et al.), disclose pharmaceutical compositions wherein recombinant IFN-.beta., IL-2 or an immunotoxin is dissolved in an aqueous carrier medium at pH 6 to 8 without the presence of a solubilizing agent. The protein is solubilized by selectively conjugating it via a coupling agent to a water-soluble polymer selected from polyethylene glycol homopolymers or polyoxyethylated polyols.
Japanese Laid-Open Patent Application (Kokai) No. 59-10524 (published Jan. 20, 1984) entitled "An Interferon Composition and a Method of Manufacturing the Same" discloses a micelle solution for rectal administration prepared by mixing (a) an unsaturated fatty acid, (b) a polyoxethylene fatty acid ester, alkyl polyoxethylene ether or sucrose fatty acid ester, (c) water and (d) interferon.
European Patent Application Publication No. 135,171 (published Mar. 27, 1985) discloses pseudomonophase, microemulsion compositions for topical application of interferons, preferably leukocyte interferon A. The compositions comprise a therapeutically effective amount of interferon, 30-70% by volume of a surface active agent having a hydrophilic-lipophilic balance (HLB) of from 12-15 and dual solubility in water/oil; 5-45% of a vegetable oil; and 5-45% water. The surface active agents disclosed therein are polyethylene glycol derivatives of castor oil composed on average of 25-36 moles of ethylene oxide per mole of castor oil. Such an oil-based microemulsion is not stable and subject to phase separation.
U.S. Pat. No. 4,507,281 discloses compositions comprising about 10.sup.4 to 10.sup.6 I.U. of human leukocyte interferon, about 1% to 5% by weight of a non-ionic surface active agent having at least one ether or amide linkage, and a physiologically acceptable carrier.
Copending, commonly owned U.S. Ser. No. 923,423, filed Oct. 27, 1986 and 100,679, filed Sept. 29, 1987, both entitled "Pharmaceutical Compositions of Recombinant Beta-Interferon and Formulation Processes" disclose and claim stable, pharmaceutical compositions of recombinant interferon-beta (IFN-.beta.) comprising as solubilizer/stabilizers one or more non-toxic biocompatible non-ionic polymeric detergents or a combination of one or more such non-ionic detergents and an additional solubilizing and/or stabilizing agent, such as, sodium dodecyl sulfate or glycerol. Said applications further disclose and claim methods of extracting IFN-.beta. from the disruptate of a host organism transformed to produce it and purifying the IFN-.beta. wherein the last purification step prior to formulation is a desalting step performed at a pH range or about 8.5 to about 10 wherein the elution buffer contains a fatty acid salt having from about 10 to about 12 carbons, and wherein the pH of the desalted IFN-.beta. pool is lowered to about 2 to about 4 thereby precipitating the fatty acid salt. The precipitated salt, preferably sodium laurate, is removed by centrifugation and filtration, and the filtrate is formulated with the non-ionic detergent containing solubilizer/stabilizers discussed therein.
There remains a need in the art for alternative formulations of biologically active, recombinant beta-interferons, preferably that are alternatives to those containing non-IFN-.beta. protein, such as albumin. The present invention meets such a need.